Pharmaceutical formulation composed of a polymer blend and an active compound for time-controlled release

ABSTRACT

A formulation containing a biologically active compound having a structure with hydrogen bonding sites blended with a first polymer having a structure with complementary hydrogen bonding sites and a second polymer that degrades to form degradation products that promote the release of the active compound from the first polymer.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority benefit under 35 U.S.C. §371 ofPCT/US01/00045 filed Jan. 2, 2001, which, in turn, claims prioritybenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/174,133 filed Dec. 31, 1999.

BACKGROUND OF THE INVENTION

The literature is replete with examples of the delayed or pulsed releaseof active agents using polymeric materials. However, it is possible todivide these systems into two basic categories; those that depend on anenvironmental stimulus to induce release of the active agent from thepolymeric matrix and those that are designed to release the drug afterparticular intervals of time have elapsed. Examples of environmentalstimuli that have been used are electrical impulses, pH or temperaturechanges, application of magnetic fields, or ultrasound.

Those systems that are time-controlled can further be divided into thosethat use a barrier technology that is placed around the active agentthat is designed to degrade or dissolve after a certain time interval,and those that use the degradation of the polymer itself to induce therelease of the active agent.

One approach has been to prepare a polymeric hydrogel composed ofderivitized dextran and to incorporate into the hydrogel, a modelprotein, I_(g)G, with an enzyme, endo-dextranase that degrades thehydrogel. It was observed that without the enzyme the release of theprotein was very slow. However, when the enzyme was included in theformulation, the release rate was dependent on the concentration of theenzyme. At high concentrations, the release was fast and complete. Atlow concentrations, the release was delayed.

Delayed release in association with hydrolytic degradation of thepolymer has also been investigated. Heller's so-called “3^(rd)generation” poly(ortho esters) are viscous ointments at room temperatureand when mixed with a model protein, lysozyme, demonstrated a delayedrelease profile. The length of the delay time was found to correlatewith polymer molecular weight and alkyl substituent of the polymer.

Ivermectin, a water insoluble antiparasitic agent for veterinaryapplications, was encapsulated in PLGA (50:50) microspheres and thesubsequent pulsed release of this agent, in vivo, was shown to bedependant on the degradation rate of the polymer matrix. Pulsed anddelayed release of active agents from PLGA microspheres was mostintensely studied by Cleland et al. The PLA or PLGA microspheres wereprocessed using a high kinematic viscosity of polymer solution and ahigh ratio of polymer to aqueous solution. This produced densemicrospheres, which required severe bulk erosion of the polymer torelease the drug. These conditions yield microspheres that have lowloading (generally 1% w/w), moderate bursts, and lag times during whichsignificant leaching of drug occurs.

SUMMARY OF THE INVENTION

The technology described in this disclosure represents a departure fromthe prior art. In this system, bonding interactions between the polymerand the active compound are used to lock the active compound into thepolymeric matrix. While one can envision several different types ofinteractions (adsorption, pi-bonding, ionic), hydrogen bondinginteractions seem to be most suitable.

Therefore, according to one aspect of the present invention, aformulation containing a biologically active compound is provided havinga structure with hydrogen bonding sites, blended with a first polymerhaving a structure with complementary hydrogen bonding sites, and asecond polymer that degrades to form degradation products that promotethe release of the active compound from the first polymer.

The formulation thus consists of three components, two polymers and abiologically active compound all blended together. The present inventionthus provides new implantable or injectable drug release systems thatrelease a pharmaceutically or biologically active compound in atime-controlled fashion, allowing the design of delay times prior torelease, the design of pulsatile release, and the design of systems withhigh loadings that are resistant to “burst” (e.g., the immediate anduncontrolled release of a substantial amount of the loaded drug within avery short initial period of use).

The present invention uses the degradation products of one polymer totrigger the release of the active compound from the other polymer. Inaddition, the delayed release of the active compound can be achievedwithout the use of barrier systems that require complex andsophisticated formulation techniques. Further, the present inventionrelies on the formation of hydrogen-bonds between the active compoundand the slow degrading, hydrophobic matrix polymer. This feature makesit possible to incorporate unexpectedly high loadings of water-solubleactive compounds into the system without any burst (as defined above).Unlike the behavior that is observed when water-soluble peptides areincorporated into any of the commonly used alpha-hydroxy acid basedpolymers such as poly(lactic acid), poly(glycolic acid) orpolydioxanone, in the system of the present invention, the formation ofhydrogen-bond mediated interactions between the polymeric matrix and theactive compound prevents burst, even at exceptionally high loadings.

There are many drugs that are more effective when given to the patientin a pulsatile manner as opposed to a continuous release fashion. Forexample, an area of great interest, currently, for this type of deliverysystem is single-shot immunization. Immunity is best induced by apulsatile delivery of the antigen, hence the need for booster shots. Ithas been suggested that it would be more economical and effective,especially in third world countries, if a delivery system for antigenssuch as tetanus toxoid or gp120 (under development for an AIDS vaccine)could be implanted once into the patient and provide for the release ofbooster doses at preprogrammed time periods.

Therefore, the present invention also includes a method for thepulsatile delivery of a biologically active compound to a patient inneed thereof by administering to the patient the formulation of thepresent invention.

This type of drug delivery is also important for hormonal based drugdelivery. Fertility and birth control drug therapy for both animals andhumans is not continuous, but rather cyclic in nature since thesetherapies work synergistically with the menstrual cycle and thecorresponding hormonal flux. This is another direction in drug deliveryin which delayed and/or pulsed release of an active compound would beapplicable.

Agricultural applications which require the timed dosing of fertilizers,weed-killers, and other active agents is another area where thisinvention would be important.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of a tyrosine-derived polyarylate,in which the arrows designate sites at which variations are introducedinto the polymer;

FIG. 2 depicts the amino acid sequence of INTEGRILIN™;

FIG. 3 depicts the release of INTEGRILIN™ from poly(DTH adipate) filmscontaining 30% (w/w) peptide;

FIG. 4 depicts the release of INTEGRILIN™ from equivalent D,L-PLA andpoly(E-caprolactone) films;

FIG. 5 depicts the percent mass retention of poly(DTH adipate) samplescontaining 30% (w/w) INTEGRILIN™;

FIG. 6 depicts the percent mass retention from equivalent D,L-PLA films;

FIG. 7 depicts percent water absorption of PCL and PLA films containing30% (w/w) INTEGRILIN™;

FIG. 8 depicts percent water absorption for an equivalent poly(DTHadipate) film;

FIG. 9 depicts percent molecular weight retention of neat poly(DTHadipate) and poly(DTH adipate) containing 30% (w/w) INTEGRILIN™;

FIG. 10 depicts the effect of ionic strength on the release ofINTEGRILIN™ (30% (w/w)) from poly(DTH adipate) films;

FIG. 11 depicts 30% (w/w) INTEGRILIN™ release from poly(DTH adipate)films at pH 2.2 without added electrolytes;

FIG. 12 depicts water uptake in 30% (w/w) INTEGRILIN™ poly(DTH adipate)films at pH 2.2 without added electrolytes;

FIG. 13 depicts the structure of poly(DTH dioxaoctanedioate);

FIG. 14 depicts the release of INTEGRILIN™ from poly(DTHdioxaoctanedioate) films;

FIG. 15 depicts the structure of poly(DTE carbonate);

FIG. 16 depicts the release of 10% (w/w) INTEGRILIN™ frompoly(DTEcoPEG);

FIG. 17 depicts the release of 15% (w/w) INTEGRILIN™ from poly(DTEcarbonate);

FIG. 18 depicts in vitro release of 30% (w/w) INTEGRILIN™ in PBS(pH=7.4, 37° C.) from D,L-PLA/poly(DTH adipate) films;

FIG. 19 depicts the chemical structure of PLGA;

FIG. 20 depicts the cumulative release of 15°/0 (w/w) INTEGRILIN™ from a50:50 blend of poly(DTH adipate) and PLGA; and

FIG. 21 depicts the NMR spectra of an INTEGRILIN™-containingPLGA/poly(DTH adipate) blend film.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The first polymer in the blend is a slowly degrading, relativelyhydrophobic and biocompatible polymer. In order to encourage theformation of hydrogen bonding interaction with the biologically activecompound, it is also necessary to choose a highly functional polymersystem as the first polymer. In its broadest embodiment, the slowdegrading, hydrophobic, biocompatible polymer can be any such polymerthat contains hydrogen-bonding sites as part of its chemical structure.In its most preferred embodiment, this slow degrading and hydrophobicpolymer is selected from the tyrosine-derived polyarylate librariesdisclosed in U.S. Pat. No. 5,216,115 and WO 99/52962, the disclosures ofboth of which are incorporated herein by reference. Members of thislibrary all share the same highly functional structural template but aredistinguished from one another by subtle structural changes. Thefunctional groups of the main template provide sites for interactions.These are pi stacking of its aromatic rings with an aromatic ring of apeptide, or hydrogen bonding of the α-amido carboxylate region with acorresponding group in the peptide. The small structural variationsbetween members allow the fine-tuning of these interactions to suitparticular proteins or peptides.

Also preferred are any of the polymers that can be derived from thetyrosine-derived diphenol compounds of U.S. Pat. No. 5,587,507 and thetyrosine-derived dihydroxy monomers of WO 98/36013, the disclosures ofboth of which are also incorporated herein by reference. In addition tothe above-referenced polyarylates, examples include the polycarbonatesof U.S. Pat. No. 5,099,060, the polyiminocarbonates of U.S. Pat. No.4,980,449, the polyphosphazenes and polyphosphates of U.S. Pat. No.5,912,225, polyurethanes, including the polyurethanes of U.S. Pat. No.5,242,997, the random poly(alkylene oxide) block copolymers of U.S. Pat.No. 5,658,995, and a wide range of other polymers that can be derivedfrom the above-referenced tyrosine-derived diphenol compounds, thetyrosine-derived dihydroxy compounds and similar peptides. All of theabove referenced patent publications are incorporated herein byreference. Notably, corresponding polymers of the tyrosine-deriveddihydroxy compounds can be made by any of the processes of any of theabove-referenced patents disclosing polymers of tyrosine-deriveddiphenol compounds.

A particularly preferred first polymer is thepoly(desaminotyrosyltyrosine hexyl ester adipate (Poly(DTH adipate)) ofFIG. 1 (y=4; R=hexyl). Poly(DTH adipate) having a weight-averagemolecular weight between about 80,000 and about 200,000 daltons isparticularly preferred.

Any biologically active moiety with hydrogen-bonding sites that can bephysically dispersed within the polymer blend can be used as abiologically active compound for release. Examples of hydrogen bondingsites include primary and secondary amines, hydroxyl groups, carboxylicacid and carboxylate groups, carbonyl (carboxyl) groups, and the like.While one can apply the current invention to any active compound thathas hydrogen bonding sites, including natural and unnatural antibiotics,cytotoxic agents and oligonucleotides, amino acid derived drugs such aspeptides and proteins seem to be most appropriate for this technology.The compositions of the present invention overcome some of thedifficulties encountered in previous attempts to formulate controlledrelease devices that show reproducible release profiles without burstand/or lag effects. In its most preferred embodiment, the activecompound is a peptide that is stable under mildly acidic conditions.

Peptide drugs suitable for formulation with the compositions of thepresent invention include natural and unnatural peptides, oligopeptides,cyclic peptides, library generated oligopeptides, polypeptides andproteins, as well as peptide mimetics and partly-peptides. Peptide drugsof particular interest include platelet aggregation inhibiting (PAI)peptides, which are antagonists of the cell surface glycoproteinIib/IIIa, thus preventing platelet aggregation, and ultimately clotformation. Preferred PAI peptides include the PAI peptides disclosed byWO 90/15620, the disclosure of which is incorporated herein byreference, particularly INTEGRILIN™ (FIG. 2), a medically useful cyclicPAI heptapeptide.

In the case of peptide drugs, interactions between the peptide and thefirst polymer inhibit the release of the peptide. These interactions arecomposed of hydrogen bonding and hydrophobic forces. It has beendiscovered that these interactions can be weakened under conditions oflow pH, resulting in the release of the peptide. Thus, one method ofachieving this is to blend in a second polymer that degrades into acidicbyproducts, into the matrix, for example, poly(glycolic acid-co-lacticacid) (PGLA). The PGLA degradation products lower the pH of the matrix,causing an interruption in the interactions and the subsequent releaseof the peptide. Control of the timing of the release can easily be doneby the choice of the initial molecular weight of this fast degradingpolymer, the copolymer ratio of lactic acid and glycolic acid within thePGLA polymer, and the choice of capping of the copolymer. Since all ofthese factors determine the kinetics of degradation, these factors canalso be used to control the release of active agents from these devices.Other useful polymers producing pH-lowering (acidic) degradationproducts include poly(glycolic acid), poly(lactic acid),polycaprolactone, poly(hydroxyalkanoic acids) such as polyhydroxybutyricacid) and poly(hydroxyvaleric acid), and the like.

It is impotant to note that the invention resides in the selection of asecond polymer that is relatively more hydrophilic than the firstpolymer. Thus, when the first polymer is highly hydrophobic, arelatively less hydrophobic polymer may be used as the second polymer,even though it might otherwise ordinarily be considered hydrophobic aswell. Likewise, when the second polymer is highly hydrophilic, arelatively less hydrophilic polymer may be used as the first polymer,even though it might otherwise ordinarily be considered hydrophilic aswell. Thus, suitable compositions may be prepared using two polymerslisted here as first polymers, or two polymers listed here as secondpolymers, provided that the first one hydrogen bonds with the activecompound, and the second one is more hydrophilic than the first anddegrades to form degradation products that promote the release of thebiologically active compound from the first polymer. One of ordinaryskill may even recognize combinations in which one of the first polymersfunctions as the second polymer and vice versa.

The compositions of the present invention are suitable for applicationswhere localized drug delivery is desired, as well as in situations wheresystemic delivery is desired. Therapeutically effective dosages may bedetermined by either in vivo or in vitro methods. For each particularcompound of the present invention, individual determinations may be madeto determine the optimal dosage required. The range of therapeuticallyeffective dosages will naturally be influenced by the route ofadministration, the therapeutic objectives, and the condition of thepatient. For the various suitable routes of administration, theabsorption efficiency must be individually determined for each drug bymethods well known in pharmacology. Accordingly, it may be necessary forthe therapist to titer the dosage and modify the route of administrationas required to obtain the optimal therapeutic effect. The determinationof effective dosage levels, that is, the dosage levels necessary toachieve the desired result, will be within the ambit of one skilled inthe art. Typically, applications of compound are commenced at lowerdosage levels, with dosage levels being increased until the desiredeffect is achieved. The release rate of the drug from the formulationsof this invention are also varied within the routine skill in the art todetermine an advantageous profile, depending on the therapeuticconditions to be treated.

A typical dosage might range from about 0.001 mg/kg to about 1000 mg/kg,preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferablyfrom about 0.10 mg/kg to about 20 mg/kg. Advantageously, the compoundsof this invention may be administered several times daily, and otherdosage regimens may also be useful.

The compositions may be administered subcutaneously, intramuscularly,colonically, rectally, nasally, orally or intraperitoneally, employing avariety of dosage forms such as suppositories, implanted pellets orsmall cylinders, aerosols, oral dosage formulations and topicalformulations, such as ointments, drops and transdermal patches.Liposomal delivery systems may also be used, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.

The following non-limiting examples set forth hereinbelow illustratecertain aspects of the invention. All parts and percentages are byweight unless otherwise noted and all temperatures are in degreesCelsius. The PAI peptide was obtained from COR Therapeutics of South SanFrancisco, Calif. Poly(DTH adipate) was prepared according to theprocedure provided in Example No. 2 of U.S. Pat. No. 5,216,115. Thepolymer used had molecular weights ranging between 80-120 kDa PEG wasobtained from Aldrich Chemicals of Milwaukee, Wis. D,L-PLA andpoly(s-caprolactone) were purchased from Medisorb and Aldrich,respectively. Both were of molecular weight 100 kDa The drug andpolymers were used without further purification. Solvents were of “HPLCgrade” and were obtained from Fisher Scientific of Pittsburgh, Pa.

EXAMPLES

INTEGRILIN™ (antithrombotic injection) was chosen as the model peptideto explore the drug delivery applications of these materials (FIG. 2).This compound is a synthetic cyclic readily water soluble heptapeptidewhich is a highly potent glycoprotein IIb/IIIa antagonist. This compoundhas successfully demonstrated antithrombogenic behavior in vivo anddevices fabricated by the formulation of this peptide into a polymermatrix with this property have many useful cardiovascular applications.In addition, this polymer contains an RGD sequence and therefore adevice containing this peptide can find application as a component inscaffolds for tissue regeneration.

The blend of INTEGRILIN™ and poly(DTH adipate) was described in U.S.Pat. No. 5,877,224. There, it was mentioned that formulating films fromthese components using the coprecipitation melt-press technique resultedin specimens that released only trace amounts of peptide when incubatedin PBS at 37° C. This was unexpected because the peptide is readilywater soluble.

Fabrication of Release Devices

Compression molded films were fabricated from a co-precipitatecontaining 30% peptide and 70% polymer by weight. This co-precipitatewas prepared by dissolving 0.15 g of peptide in 5 ml of methanol (HPLCgrade) and 0.35 g polymer in 5 ml of methylene chloride (HPLC grade) andmixing the two solutions together to form a clear solution. Thisresultant solution was added drop-wise into 100 ml of stirred ethylether maintained at −78° C. White spongy precipitates were formed,filtered using a sintered glass filter, and dried under vacuum. Afterdrying the co-precipitate was compression molded at 90° C. under apressure of 5,000 psi. Films with a thickness of 0.1 mm (±0.02 mm) wereobtained.

Device Characterization

Peptide loading was determined by dissolving 10.0 mg of a film in THF(HPLC grade) (1.0 ml) in a 10 ml volumetric flask and adding PBS(phosphate buffer saline) until the 10 ml line. The mixture was stirredfor a minimum of 6 hours followed by HPLC analysis of the drug contentin the aqueous medium. Methylene chloride replaced THF whencharacterizing samples composed of PLA or poly(s-caprolactone) due totheir insolubility in THF.

Peptide Release Study

Films were cut into 0.5 cm² squares. The mean mass of the samples was 21mg (±5). Each specimen was individually placed into 20 ml glassscintillation vials containing 10 ml of phosphate buffered saline (pH7.4, 37° C.). The standard PBS solution used was composed of 10 mMphosphate buffer saline, 138 mM NaCl and 2.7 mM KCI. The buffer waschanged at each time point and analyzed by HPLC for release of thepeptide. There was a minimum of three samples per time point, eachsample originating from a different film. The HPLC method involved a 3cm C-18 Perkin Elmer cartridge column with a gradient mobile phase whichbegan at 80% water/20% acetonitrile and ended with 75% water during aperiod of 5 minutes at a flow rate of 1 ml/min. Both the acetonitrileand water contained 0.1% (v/v) trifluoroacetic acid. The column wascalibrated with known concentrations of the peptide dissolved in PBS toestablish a calibration curve and the INTEGRILIN™ contained in thebuffer of each sample was quantified using this curve. The HPLC pumpused was a Perkin Elmer Series 410 LC pump and the detector used was aPE LC-235 diode array UV-VIS detector set at 280 nm. The data collectedwas analyzed using a PE Nelson 3000 Series Chromatography Data System.

At designated times, the samples were removed, rinsed with deionizedwater, blotted with a Kimwipe tissue and either placed in a vial forsubsequent vacuum drying for mass retention and molecular weightretention studies or used for thermal gravimetnc analysis (TGA) wateruptake studies. Those devices that were not needed for gel permeationchromatography (GPC) or TGA studies were dissolved in organic solventsubsequent to drying and the peptide content extracted to ensure thatall loaded peptide was accounted for.

Water Content Determination using Thermogravimetric Analysis (TGA)

The quantity of water absorbed by the incubated samples was measuredusing a TGA 951 (TA Instruments, Inc.). The sample was removed from thebuffer, rinsed in deionized water to remove the buffer salts, andblotted dry with a Kimwipe tissue. A small sample (10 mg) was cut fromthe specimen and placed in an aluminum TGA pan. The sample was heatedunder a nitrogen flow at a rate of 10° C./min from room temperature to225° C. The water uptake was measured by the loss in weight of thesample as it was heated from room temperature until 150° C.

Water Content Determination Using the Microbalance

At pre-determined time points, the samples were removed from the buffer,rinsed with deionized water and blotted dry. The sample's wet weight(W_(w)) was immediately taken using an electronic balance. The dryweight (W_(d)) was taken after the sample was dried under vacuum for atleast two weeks, by this time constant weight was achieved. The amountof water uptake was calculated from the following equation:% Water uptake=[(W _(w) ,−W _(d))/W _(d)]×100Differential Scanning Calorimetry Analysis (DSC) to Measure the MeltingPoint of the Peptide and Melt Transitions in the Polymer Film

DSC was used to determine the melting point of the peptide. A sample ofapproximately 2 mg of peptide was weighed out and sealed in a crimpedaluminum DSC pan. The sample was heated at 12° C./min from roomtemperature to 200° C., under nitrogen flow. DSC was also used todetermine whether there is a melting transition associated with thepolymer films that contain 30% (w/w) peptide. A sample size of 6 mg offilm was sealed in a crimped aluminum DSC pan and heated at 12° C./minuntil 200° C., under nitrogen flow. The melting point of the sample wasdetermined by the temperature at which the sharp endotherm of meltingoccurred. All data was analyzed using the first-run thermogram. An emptyaluminum pan was used as a reference in each experiment. The particularinstrument used was a DS C 910 (TA instruments) and the instrument wascalibrated with indium (m.p.=156.61° C.) before use.

Percent Mass Retention Study

The percent mass retention of the samples was calculated in thefollowing manner. The sample was removed from the PBS incubation medium,rinsed in deionized water, and blotted with a Kimwipe tissue. It wasplaced in a fresh vial and dried under vacuum for 2 weeks. Followingthis dessication period, it was weighed (W_(d)). The mass obtainedfollowing incubation and drying was compared to the initial mass(W_(o)). The formula for calculating percent mass retention is thefollowing:% Mass loss=[(W _(o) −W _(d) /W _(o)]×100Molecular Weight Determination of the Polymers Using GPC

Film samples were dissolved in THF to obtain a concentration of 5 mg/mland prefiltered through a 25 um glass fiber filter and subsequentlyfiltered through a 0.45 um PTFE filter prior to injection into the GPC.The molecular weights of the poly(DTH adipate) samples were calculatedrelative to a set of monodispersed polystyrene standards (PolymerLaboratories, Ltd. Church Station, U.K.) without further corrections.The GPC chromatographic system consisted of a Waters 510 HPLC pump, aWaters 410 differential refractometer detector, and a Digital Venturi's466 PC running Millenium (Waters Corp.) software for data processing.Two PL-gel columns 30 cm in length (pore sizes of 10³ and 10⁵ A; PolymerLaboratories LTD, England) operated in series at a flow rate of 1 ml/minin THF. Samples composed of PLA or PCL were dissolved in methylenechloride instead of THF, but otherwise analyzed in the same way as thepoly(DTH adipate) samples.

Formulation of Poly(DTH adipate)/PLA Blend Films

Release devices composed of D,L-PLA and poly(DTH adipate) blendscontained 30% (w/w) peptide. In the case of the 50/50 D,L-PLA/poly(DTHadipate), 0.15 g of INTEGRILIN™ was dissolved in 5 ml of methanol and0.175 g of PLA and 0.175 g of poly(DTH adipate) were dissolved in 2.5 mlof methylene chloride, respectively. All the solutions were combined toform a clear solution. From this point the procedure for the fabricationof the poly(DTH adipate) devices was followed.

Devices composed of 75/25 PLA/poly(DTH adipate) were fabricated bydissolving 0.15 g of INTEGRILIN™ into 5 ml of methanol, 0.26 g ofD,L-PLA in 4 ml of methylene chloride and 0.09 g of poly(DTH adipate) in2 ml of methylene chloride and combining the three solutions. From thispoint the procedure for the fabrication of the poly(DTH adipate) deviceswas followed.

Fabrication and Incubation of Films Under Acidic Conditions

The same formulation protocol mentioned above was followed for thesefilms, with the exception that concentrated HCl (12 molar) was addeddrop-wise to the stirred peptide/methanol solution until the pH, asmeasured by a pH meter dropped from 6.8 to 2.

The acidic media for the in vitro incubation studies conducted at pH of2 was prepared in the following manner. Standard PBS solution was usedand 12 M HCl was added drop-wise into the PBS until the PBS until the pHmeter indicated that the desired pH had been obtained.

Incubation of Films Under Varying Ionic Strength Conditions

Three sets of films were prepared in the standard method mentionedabove, one set was incubated in HPLC water, used as is. Another wasincubated in the standard PBS buffer. The last set was incubated in PBSbuffer that was twice the concentration of the standard PBS solution.

The Effect of the Peptide on the Glass Transition Temperature ofPoly(DTH adipate)

The glass transition temperature of sets of films was measured usingDynamic Mechanical Analysis (DMA). Measurements were performed on a DMA983 from TA Instruments in a flexural bending deformation mode ofstrain. Each set of films contained a different weight percentage ofpeptide ranging from 0%-30% (w/w) of peptide. Samples of approximatesize 5×10×1 mm were cut from the films and mounted on the instrumentusing low mass clamps, after calibrating the instrument with the lowmass clamps. The samples were cooled using a liquid nitrogen coolingaccessory to −30° C. and heated at a rate of 4° C./min until 70° C. Thefrequency was fixed at 1 Hz and the amplitude was 1 mm. The glasstransition was read from the maxima of the E″ peak.

Formulation of INTEGRILIN™/PLGA/poly(DTH adipate) Blend Films

Devices composed of INTEGRILIN™/PLGA/poly(DTH adipate) were prepared bythe dissolution of 0.075 g of peptide in 2 ml of methanol, 0.21 g ofPLGA in 3 ml of methylene chloride, and 0.21 g of poly(DTH adipate) in 3ml of methylene chloride. The PLGA solution was pipetted into thepoly(DTH adipate) solution. The INTEGRILIN™ solution was pipetted intothe mixed polymer solution. The clear solution was added drop-wise intocold diethyl ether (−78° C.). The remainder of the procedure is the sameas described above.

Characterization of Blend Films

The peptide loading of the film was determined as described above, usingmethylene chloride to dissolve the polymer matrix instead of THF.

The ratio of PLGA to poly(DTH adipate) in the polymer blend films wascharacterized using nuclear magnetic resonance (NMR)(FIG. 21). For eachfilm, the ratio of the integration of the PGA methylene protons to thatof the PLA methine proton was compared to the theoretical. It wasdetermined that the experimental and theoretical ratios were within 10%of each other. Next, the integration of the PGA methylene protons (4.8ppm) was compared to the integration of the methylene protons of thepoly(DTH adipate) at 4.1 ppm. This peak at 4.1 ppm is associated withthe protons on the methylene group that is next to the oxygen of theester in the pendent chain. The theoretical ratio of these PGA protonsto the above mentioned protons of poly(DTH adipate) is 3.86. All filmswere characterized in this manner and in all cases the error of theratio of PGA to poly(DTH adipate) was within the error of the ratio ofthe PGA to the PLA.

For the NMR analysis 20 mg of the film was dissolved in 0.75 ml ofdeuterated chloroform. This solvent was chosen since the peptide is notsoluble in it and it would therefore be transparent to the NMR, therebypreventing unnecessary complications of the spectra. The samples wereanalyzed using a Varian 200 MHz instrument. Integrations were made onthe spectra after 256 acquisitions. Integrations on the samples wererepeated 3 times to ensure accuracy, and values for the integrationswere taken from the average of the three values.

Characterization of Film Surfaces of Using SEM

Following rinsing and drying under vacuum samples were attached to a SEMstub and sputter coated in a Balzers SCD 004 sputter coater with 7 rimof gold palladium. They were observed in an Amray 18301 SEM at 20 kV,magnification=90×.

Determination of Miscibility of Polymer Blends Using DSC

A Thermal Analysis 2100 system incorporating a DSC 910 (TA Instruments,DE) was employed, and calibrated with indium (m.p. 156.61° C.) beforeuse. A polymer with a known weight (˜4 mg) was sealed between twocrimped aluminum pans. The sample was subjected to two consecutiveheating scans in order to ensure an identical thermal history for allsamples. In the first scan, the sample was heated at the rate of 10°C./mm to 110° C. After the temperature was kept isothermal for 10minutes, the sample was cooled to −20° C. using liquid nitrogen. Thisstep erases the thermal history of the sample. The second scan wasperformed immediately thereafter at the rate of 10° C./min. The glasstransition temperature was determined in the second scan as the midpointof the endothermic step transition associated with the glass transition.

Formulation of INTEGRILIN™ with Poly(DTH adipate)

Films composed of poly(DTH adipate) containing loadings of 5, 10, 15,20, and 30% (w/w) peptide were prepared. Films containing even thehighest loading were clear and flexible. In contrast, the films composedeither of D,L-PLA or poly(E-caprolactone) (PCL) containing the same loadof peptide were opaque and brittle. The clarity of thepeptide/polyarylate films indicated that the phase separation in thecase of the peptide and poly(DTH adipate) was sufficiently reduced thatthe separate polymer and peptide domains were too small to scatterlight. This demonstrated an enhanced compatibility of peptide andtyrosine-derived polymer relative to blends of D,L-PLA and peptide orPCL and peptide.

The flexibility of the polyarylate films that contained peptide relativeto those composed of the peptide and either of aliphatic polyesters canbe explained by the lower glass transition temperature of thepolyarylate (37° C.) as compared to that of PLA (52° C.), and theamorphous nature of the polyarylate as compared to PCL.

Release of Peptide from Films Incubated at 37° C. and at pH=7.4

In this experiments the in vitro release behavior of the peptide, undersimulated physiological conditions, from various polymer matrices wasobserved. Unexpectedly, both the aliphatic polymers released the peptidecompletely within three hours. In contrast, the poly(DTH adipate)demonstrated only trace release, over a period of 77 days, under theidentical conditions (FIGS. 3 & 4).

Percent Mass Retention of Incubated Samples

Poly(DTH adipate) samples containing 30% (w/w) peptide lost on average5% mass during the 77 day incubation period (FIG. 5). In contrast, theD,L-PLA samples that were formulated in the identical fashion as thepoly(DTH adipate) samples lost about 30% of their mass within two hours(FIG. 6). The results of these experiments, therefore, were consistentwith the data obtained from the HPLC. In the case of the poly(DTHadipate) films containing 30% (w/w) peptide, the HPLC data indicate thatthese films released-less than 10% of the loaded peptide (FIG. 3). Thistranslates into a mass loss for the entire sample of about 3% over the77 day period. This is in agreement with the average 5°/o mass lossobserved for these samples.

In contrast to the poly(DTH adipate) samples that demonstrated minimalmass loss, the PLA samples showed extensive mass loss. These filmsamples also contained 30% (w/w) peptide. HPLC data indicated that thesesamples released all of the peptide that they contained, whichtranslates into a 30% mass loss over the three hour incubation period.The resulting percent mass retention data is about 70% for these samplesis therefore in agreement with the HPLC results. Furthermore, since thepeptide was released so rapidly by the PLA and PCL matrices, it can beconcluded that the peptide is small enough to readily diffuse throughthe polymer chains and the development of pore structures andinterconnecting channels is not necessary to release the molecules ofpeptide that are deep within the film. Therefore there should be minimalimpedance for release of the peptide from the polyarylate.

Measurement of Water Absorption by Polymer Films During Incubation

Specimens of poly(DTH adipate) containing 30% (w/w) peptide absorbedabout 10% by weight water within the first day and maintained that levelof swelling throughout the entire incubation period. Also, the presenceof the peptide increased the water absorption of the polymer from about3% by weight to 10% by weight (FIG. 8). Samples of PLA and PCLcontaining identical loading of peptide to the poly(DTH adipate) alsoabsorbed water within that range during the 2-3 hours that they wereincubated (FIG. 7).

The similarity in water uptake between the three polymers whenformulated in an identical manner with the peptide indicates that theabsorption of water is not the determining factor for release of thepeptide.

The Effect of the Peptide on the Molecular Weight of the Polymer

One of the amino acid residues on the peptide is an aspartic acid.Aspartic acid is a moiety that could introduce acidity into the polymerwhen the polymer is blended with the peptide. Consequently, aninvestigation of the molecular weight degradation of the polymer wasmade and compared to rate of degradation for the neat poly(DTH adipate)(FIG. 9).

As an additional control, samples composed of a blend of 10% (w/w) PEGand 90% (w/w) poly(DTH adipate) were included in these studies becausethese samples absorb 20% by weight water as measured by the TGA. Thisrepresents more water than is absorbed by the polymer samples containing30% (w/w) peptide and this therefore can function as a control for theeffect of the added water on the molecular weight degradation of thepolymer. The results of these studies were that the samples containingpeptide did degrade at a faster rate than the samples that did notcontain peptide. After a period of over 2 months the poly(DTH adipate)samples containing 30% (w/w) peptide had undergone 40% molecular weightdegradation. In contrast, those samples without peptide demonstratedalmost no degradation during this time period.

In addition, the increased amount of water in the polymer matrix did notaffect the rate of molecular degradation at all. There did not appear tobe any significant difference in the rate of molecular weightdegradation between the poly(DTH adipate) samples containing PEG and theneat samples. Therefore, it can be concluded that it was the presence ofthe peptide that had the catalytic effect on the degradation of thepolymer. However, this increase in degradation rate was not significantenough to affect the release of the peptide.

Effect of Ionic Strength of the Medium on the Release of the Peptide

Poly(DTH adipate) films containing 30% (w/w) peptide were prepared inthe standard manner. The pH of the incubation media remained about 7,but the ionic strength of the release media was varied. The in vitrorelease of the peptide in HPLC water, in the standard PBS solution (10mM phosphate buffer saline, 138 mM NaCl, 2.7 mM KCl), and in PBS bufferformulated at twice the concentration (20 mM phosphate buffer saline,276 mM NaCl, 5.4 mM KCl) was measured and compared (FIG. 10). It wasobserved that the rate of release of peptide was four times greater inHPLC water as compared to the release rate in phosphate buffer. Theseresults demonstrate some hydrophobic interactions between the peptideand polymer, such as the pi stacking of the tryptophan ring of thepeptide with the phenolic ring of the polymer.

Incubation of Poly(DTH adipate) Films Containing 30% (w/w) Peptide inAcidic and Low Ionic Strength Conditions

Samples containing 30% (w/w) peptide were prepared under standardconditions and incubated in HPLC water containing 0.1% (v/v)trifluoroacetic acid, the pH of which was 2.2. The release rate of thepeptide, when both pH and ionic strength of the incubation media werelowered was greater (FIG. 11) than when just one factor was lowered.When just the pH was lowered, 12% of the loaded peptide was releasedwithin three days. When just the ionic strength was reduced 8% of theloaded peptide was released within three days. When both parameters werelowered simultaneously 20% of the loaded peptide was released withinthis time period. Despite enhanced release in these conditions, thepeptide was not “dumped out” as in the case of D,L-PLA but there was acontinuous diffusion of the peptide from the poly(DTH adipate) matrix.

However, what was unexpected was the absorption of water under theseconditions (FIG. 12). Within the first day of incubation these samplesswelled three times relative to the samples incubated in the standardPBS solution, and by the seventh day these samples swelled by seventimes. From FIG. 9, it can be determined that the neat polymer, byitself, does not increase its absorption of water during this initial 7day time period when incubated in the standard PBS solution. Moreover,since this polymer is relatively hydrophobic, it would not be expectedthat the change in incubation conditions would promote such an increasein the percentage of water uptake by the neat polymer. Therefore, it canbe inferred that it is the peptide within the matrix that is the sourceof this large water uptake.

Therefore, incubation in the standard PBS solution favors theinteraction of the peptide with poly(DTH adipate) rather than withwater, hence, there was no increase in swelling beyond the initial 10%even over many weeks of incubation in these conditions. However, inconditions where the peptide-polymer interactions are weakened, as inthis case, where both the pH and ionic strength of the incubation mediawere lowered, there is more of a driving force for the peptide tointeract with water and consequently, there was a steady increase in theswelling of the film as more peptide molecules were exposed to andinteracted with water.

Under these conditions of increased acidity and lowered ionic strengththe film samples also turned opaque immediately. This opaqueness, notedonly under the circumstances where there was enhanced release of thepeptide from the poly(DTH adipate) films, appears to be correlated withincreased water absorption by the film samples. The weakening of theintensity of the peptide-polymer interactions result in an increase inwater absorption and the developing opacity, is caused by the water thatoccupies the free volume within the polymer matrix.

The absorption of 10% by weight water was sufficient to release thepeptide to completion in the case of the aliphatic polymers. However,samples whose matrix was composed of poly(DTH adipate) instead of PLA,absorbed 70% by weight water and yet did not release the peptide in thesame “dumping” manner that the PLA and PCL matrices did at 10% by weightof water absorption.

The interaction of the peptide with the tyrosine-derived polyarylatearises from the unique structure of the polymer in which the amide bondof each repeat unit is in close proximity to the pendent ester in thesame unit. This entire region can be considered as one functional group,the α-amidocarboxylate group, and can act as a pocket for the hydrogenbonding of various groups on the peptide.

Peptide-polymer Interactions with Other Tyrosine-Derived Polymers

Several other polymers were screened for the diffusion of the peptide.The loadings of peptide used in these screening experiment were lowerthan those used with poly(DTH adipate), but they were sufficient toexpect release of this readily water-soluble peptide barring anyinteractions to impede it.

Poly(DTH dioxaoctanedioate) was the first alternate but structurallyrelated polymer investigated. This polymer contains the DTH repeat unit,which makes it similar to poly(DTH adipate). However, this polymer issynthesized by polymerizing DTH with dioxaoctanedioic acid (FIG. 13)instead of adipic acid.

No peptide was released from these films (FIG. 14) indicating thatincreasing the hydrophilicity of the polymer does not have an effect onthe release of the peptide. The water uptake of the films was alsomeasured and found to be 5% by weight in the case of films thatcontained 10% (w/w) peptide and 10% by weight in the case of filmscontaining 20% (w/w) peptide. This indicates that although the loadingof the peptide is lower in these specimens there is the same amount ofwater in bulk in poly(DTH adipate) specimens containing 30% (w/w)peptide as in poly(DTH dioxaoctanedioate) containing 20% (w/w) peptide.In addition, the structure of this polymer differs from poly(DTHadipate) only in the structure of the flexible backbone unit. Since therelease behavior of this polymer is similar to that of poly(DTHadipate), and the structural differences between the two polymers lieonly in the structure of the backbone unit, it can be concluded that theDTH unit is what is most integral to the peptide-polymer interactions.

Another polymer structure was substituted for poly(DTH adipate),poly(DTE_(0.95)-co-PEG_((1000)0.05) carbonate). This polymer is a randomcopolymer of desaminotyrosyl tyrosine ethyl ester (DTE) andpoly(ethylene glycol) (PEG) (FIG. 15) and shares the basicdesaminotyrosyl tyrosine alkyl ester repeat unit with the poly(DTHadipate), but contains carbonate linkages and not ester in the backbone,and no diacid component. The absence of the diacid component and thesimilarity in the tyrosine-derived repeat unit should further confirmthat it is the tyrosine-derived component and not the diacid that isinvolved in these interactions should the peptide fail to diffuse fromthis polymer. Films containing 10% (w/w) peptide were prepared.

Peptide release from these polymers also was minimal (FIG. 16). Thewater uptake of these samples was also measured, and during the periodof incubation the film samples absorbed 10% by weight water. Again, thisis the same amount of water absorbed by the PLA, PCL, and poly(DTHadipate) samples. Although the peptide loading is lower in these filmsit is not surprising that the water uptake is as much as samples ofthese other polymer systems since the PEG increases the hydrophilicityof these samples. These data demonstrate that the minimal release of thepeptide from polymers containing the DTR unit is the result of thetyrosine-derived repeat unit being the structure responsible for theabsence of diffusion of the peptide from the polymer.

Poly(DTE carbonate) was also formulated with 15% (w/w) peptide. Thispolymer structure contains only the desaminotyrosyltyrosine ethyl esterwith carbonate linkages and does not contain any PEG. These films alsoshowed the same behavior as the tyrosine-derived polyarylates (FIG. 17).The water uptake of these films was also measured and found to be 6% byweight over the incubation period.

In Vitro Release of the Peptide from Poly(DTH adipate)/D,L-PLA Blends

The in vitro release study of the peptide from poly(DTH adipate)resulted in about 5% release of the loaded peptide. Release of thepeptide from D,L-PLA matrices under the same conditions resulted in thecomplete release of the loaded peptide within three hours. In vitrorelease studies of the peptide from the D,L-PLA/poly(DTH adipate) blendfilms resulted in a moderate burst and an extended release of thepeptide relative to release from D,L-PLA (FIG. 18). Those samples with a1:1 ratio of poly(DTH adipate) to D,L-PLA released 18% of the totalpeptide load within five days. Those samples containing a 3:1 ratio ofD,L-PLA to poly(DTH adipate) released 40% of the peptide load withinfive days. In both formulations, therefore, the initial burst of peptidewas reduced relative to the burst from the D,L-PLA films. Moreover, therelease was extended from the original three hours associated with theD,L-PLA matrices.

Pulsatile Delivery of a Model Water Soluble Peptide:

Preparation of Films Composed of Peptide, PLGA, and Poly(DTH adipate)

TABLE 1 Characteristics of PLGA in each film set Film Set I II IIIM_(w), (kilodaltons) 12 25 62 Polydispersity 2.4 3.4 2.0 T_(g) (° C.)39.5 45.3 48.2 PLA:PGA (mole ratio) 53:47 52:48 54:46

PLGA is a resorbable copolymer composed of poly(lactic acid) andpoly(glycolic acid) that is known to release acidic compounds during thedegradation process (FIG. 19). Three sets of blend films were prepared.All films contained 15% (±2) (w/w) peptide, 42% (w/w) uncapped PLGA, and43% poly(DTH adipate). They only differed in the molecular weight ofPLGA that they contained (Table 1).

Visual Inspection of Blend Films

In contrast to the transparent nature of the peptide/poly(DTH adipate)films, these films appeared homogeneously opaque, very similar to theblends composed of peptide, D,L-PLA and poly(DTH adipate) describedpreviously, suggesting increased phase separation of thepeptide/PLGA/poly(DTH adipate) relative to the peptide/poly(DTH adiapte)films. In addition, these films were less flexible as compared to filmsmade without PLGA. As expected, the brittleness increased withdecreasing molecular weight of PLGA.

Differential Scanning Calorimetry of Blend Films

Two glass transition temperatures were evident on the thermograms of allfilms containing the blend of PLGA and poly(DTH adipate) confirming thephase separation present in these blend films. The lower T_(g) occurringat about 34° C. is associated with the poly(DTH adipate) domains. Thehigher one at 49° C. corresponds to the PLGA domains. The appearance ofthe two glass transitions signifies that the two polymers are immiscibleand therefore, there are separate domains of PLGA and poly(DTH adipate)present in the films.

It was also noted in samples that were heated to 200° C. that there wasno endotherm in the corresponding thermogram, characteristic of the neatpeptide, indicating that in these matrices, too, the peptide is notpresent in discrete crystalline domains.

Release of the Peptide from Poly(DTH adipate)/PLGA Blend Film Matrices

Film samples of the PLGA and poly(DTH adipate) blends were incubated inPBS at 37° C. (FIG. 20). As expected, there was a small burst. The sizeof this burst appears to be related to the molecular weight of the PLGA.In general, samples containing the lowest molecular weights of PLGA wereassociated with larger bursts (11% -18% of loaded peptide) than samplescontaining the highest molecular weight of PLGA (6% of the loadedpeptide). However, no sample released more than 20% (w/w) of the loadedpeptide in this fashion. The source of this correlation betweendecreasing molecular weight and burst, perhaps, is that the lowermolecular weight polymer is more hydrophilic than the higher molecularweight polymer, due to the larger number of endgroups present in thelower molecular weight polymers as compared to higher molecular weightpolymers. Therefore, the lower molecular weight polymers have more of anaffinity for the peptide. As the molecular weight increases, thehydrophilicity decreases, resulting in a smaller initial release.

The lag time between release phases also displayed a direct relationwith the initial molecular weight of the PLGA. There was a lag time ofless than 5 days associated with the films containing the lowestmolecular weight of PLGA. Furthermore, the release of the peptidefollowing the lag phase was quite rapid. The entire load of peptide wasreleased by twelve days, and the majority within about four days.

Samples containing PLGA of molecular weight 25 kDa displayed a lag timethat varied from 18 to 26 days (the majority of the samples exhibited adelay time of approximately 18 days, and only one sample displayed a 26day lag time). The release of peptide after the lag phase was also quiterapid and the entire load of peptide was released within about 5 days.

Samples containing the highest molecular weight PLGA (63 kDa) showed alag time that varied from 27 to 34 days, with most of the samplesconcentrated around the 27 day mark. After the lag time the release wasrapid, but not as rapid as samples with lower molecular weight PLGA.These samples released their load of peptide over a period of about 10days.

During the lag periods, less than 2% of the loaded peptide was leachedout from the blend films. This signifies that although this formulationdoes not contain a true barrier but relies only on peptide-polymerinteractions to prevent release of the peptide during the delay phase,this system works just as well as a barrier technique. As a control,samples of poly(DTH adipate) containing 15% (w/w) peptide but no PLGAwere formulated. These samples released almost no peptide during theincubation period.

The time required for the PLGA to degrade sufficiently to form theacidic byproducts controls the length of lag time. Samples with lowerinitial molecular weight of the PLGA require a shorter length of time toreach the critical degradation phase. Therefore, those samples with thelowest molecular weight of PLGA were associated with the shortest lagtimes and those with the largest initial molecular weight correspondedto the longest lag time before release. The samples containing thelargest initial molecular weight of PLGA also displayed the slowest rateof release following the lag time. Although there is a sufficientconcentration of acid products in the matrix of the polymer to releasethe peptide, the concentration did not quite reach the same level asthat of the samples from polymers with lower initial molecular weights.Therefore, the effect of weakening the interactions is less intenseresulting in a slower release of the peptide

Monitoring the pH of the Incubation Media

During the in vitro peptide release experiment the pH of the incubationmedia was monitored at each time point during the buffer change. As acontrol, a poly(DTH adipate) film containing 15% (w/w) INTEGRILIN™ wasincubated and its pH measured, as well.

Samples of PLGA,_(12,000) maintained a pH of 7.4 for 9 days. Followingthis period the pH of the buffers of these samples steadily fell and bythe end of the incubation period the pH of the buffers had reached 7.0.The sharpest decline in pH exactly mirrored the sharpest increase inrelease of the peptide.

The pH of the buffers of the samples of PLGA_(25,000) measured 7.4 forthe first 15 days of incubation. Following this period the pH of thebuffer was 7.2 and this pH was maintained until 21 days when the bufferreached its lowest pH of 6.9. For the remainder of the incubation periodthe pH of the buffer vacillated between 7.0 and 7.2.

The pH of the buffers of the samples containing the highest molecularweight PLGA, maintained physiological pH until the 32^(nd) day. At thispoint the pH dropped to 7.0 and for the remainder of the incubationperiod the pH of the buffer vacillated between 7.0 and 7.3.

The coinciding of the drop in the pH of the buffer with the release ofthe peptide occurred because during the incubation of the samples thePLGA phase in the films began the molecular weight degradation process,and the acidic degradation products accumulated within the bulk of thematrix. Eventually a critical acid concentration was reached and thepeptide-polymer interactions were weakened, resulting in the release ofthe peptide. However, the water-soluble degradation products alsodiffuse out of the matrix, so there is a codiffusion of the peptide withthe acidic degradation products. Because of this codiffusion effect, thedrop in pH of the buffer either coincides with the release of thepeptide or occurs within a short time after the release of the peptidehas begun.

Percent Mass Retention of Blend Films.

The samples composed of peptide, PLGA, and poly(DTH adipate) blends wererinsed after incubation, dried, and weighed after drying. The percentmass retention of these samples all ranged between 60 and 70% (Table 2).If only the peptide was released the percent mass retention would beapproximately 85%. The fact that the mass loss was greater than 15%suggests that some degradation occurred. Since the films containing justpeptide and poly(DTH adipate) did not show significant mass loss, thecomponent lost from these blends was PLGA which degrades into watersoluble oligomers.

Percent Mass Retention of Blend Films

TABLE 2 Percent mass retention of film samples containing a PLGA -poly(DTH adipate) blend MW of PLGA in film set Percent Mass RetentionDays Incubated 12 kda 70 ± 3 12 25 kda 60 ± 4 35 62 kda 62 ± 4 50 NoPLGA   99 ± 0.2 44

The molecular weight of the poly(DTH adipate) in these blend samples wasalso investigated. After drying, film samples were analyzed, using GPC,for their molecular weight. As a control, poly(DTH adipate) filmscontaining 15% (w/w) peptide were also incubated in PBS and the percentmolecular weight retention was compared to those samples that containedPLGA. To calculate the percent molecular weight retention, the molecularweights of the films after incubation were normalized to the molecularweight of the virgin poly(DTH adipate) (100 kDa) that was used toprepare the films. The virgin polymer was used and not the molecularweight of the films before incubation as would normally be done becausethe blended PLGA can dramatically alter the true value for the molecularweight of poly(DTH adipate) by artificially raising the proportion oflow molecular weight fractions. The effect is more dramatic before thefilms are incubated than after since during incubation the PLGA isreduced to oligomers and the molecular weight data represented by themain peak in the GPC trace of the film after incubation, for the mostpart, does not include the molecular weight of PLGA or includes areduced fraction of PLGA as compared to before incubation. Consequently,by normalizing the data to the virgin polymer the change in molecularweight of poly(DTH adipate) during incubation is followed and not thechange in molecular weight of the PLGA blend (Table 3).

TABLE 3 Percent Mw Retention following poly(DTH adipate) incubation inPLGA film samples c Percent Molecular Weight PLGA Mw in film setRetention of poly(DTH adipate) Days Incubated 12 kda  53 ± 12 16 25 kda47 ± 5 38 62 kda 40 ± 3 47 No PLGA 52 ± 1 45

No correlation between the percent molecular weight retention of theblend films and release of the peptide was observed (Table 3). Thesample sets containing the two lower molecular weight polymers of PLGAdegraded to the same extent as the control film containing no PLGA.However, the films containing PLGA released the peptide and the controldid not. Therefore, a massive degradation of poly(DTH adipate) did notoccur. This is not the mechanism responsible for the release of thepeptide

Scanning Electron Microscopy (SEM) of Blend Film Surfaces FollowingIncubation

Changes in surface morphology during incubation of poly(DTHadipate)/PLGA_(12,000) films containing 15% (w/w) peptide wereinvestigated using SEM. A comparison of surface morphology before andafter incubation showed that the surface, originally, was relativelysmooth, although quite porous. After an incubation period of 16 days,the surface became much rougher eliminating any resemblance to thesmoothness that was observed before incubation. In addition, largecracks and holes developed. These pores, holes and cracks are PLGA-richdomains that are now empty due to the degradation of the PLGA and thesubsequent dissolution of the water-soluble degradation products. Thesedomains do not represent peptide-rich domains, because such phaseseparation would have led to a fast and immediate release of the peptideand not this delayed response.

The surface of control samples composed of 15% (w/w) peptide and 85%(w/w) poly(DTH adipate), in contrast, did not appear to changesignificantly over the same 16 day incubation period. This furtherconfirms that these samples did not undergo the same dramatic changesthat the samples containing PLGA did.

The polymer blends of the present invention thus provide formulationsfor the pulsatile release of biologically active compounds in which theactive compound is “locked” by means of hydrogen bonding in a relativelyhydrophobic polymer matrix until such time as the less hydrophobicpolymer of the blend hydrolytically degrades to promote the release ofthe active compound from the more hydrophobic polymer. The length ofdelay and the rate of delivery following the delay can be reproduciblycontrolled through the selection of materials and the quantitiesemployed.

The foregoing examples and description of the preferred embodimentshould be taken as illustrating, rather than as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not to be regarded as a departure fromthe spirit and scope of the invention, and all such modifications areintended to be included within the scope of the following claims.

1. A formulation comprising a biologically active compound having achemical structure with hydrogen bonding sites, a first biocompatible,hydrolytically degrading polyarylate with hydrogen bonding sitescomprising tyrosine-derived diphenol monomer units, and a secondbiocompatible polymer that is less hydrophobic than said polyarylate, sothat said second polymer degrades hydrolytically to form acidicdegradation products that promote the release of said active compound,and wherein said second polymer is selected from the group consisting ofa second polyarylate comprising tyrosine-derived diphenol monomer units,a poly(glycolic acid), a poly(lactic acid), a poly(glvcolicacid-co-lactic acid) (PGLA), a polycaprolactone and apoly(hydroxyalkanoic acid).
 2. The formulation of claim 1, wherein saidbiologically active compound is pharmaceutically active.
 3. Theformulation of claim 2, wherein said pharmaceutically active compound isa peptide.
 4. The formulation of claim 3, wherein said peptide is aplatelet aggregation inhibiting (PAI) peptide.
 5. The formulation ofclaim 4, wherein said PAI peptide is a synthetic cyclic heptapeptideantagonist of glycoprotein IIb/IIa.
 6. The formulation of claim 1,wherein said polyarylate is randomly block copolymerized with apoly(alkylene oxide).
 7. The formulation of claim 1, wherein saidpolyarylate is poly(DTH adipate).
 8. The formulation of claim 1, whereinsaid second polymer is PGLA.
 9. The formulation of claim 7, wherein saidsecond polymer is PGLA and said active compound is a pharmaceuticallyactive peptide.
 10. A method for the pulsatile delivery of an activecompound to a patient in need thereof comprising administering to saidpatient the formulation of claim 1.